Systematic efforts to identify and understand protein function and structure have been facilitated by miniaturized assays that use extremely low sample volumes, yet allow for rapid and simultaneous analysis of thousands of proteins. Such “high through-put” miniaturized technology was first developed for studying nucleotide sequences. While much of the methodology used to design and prepare oligonucleotide arrays can be used with proteins, certain protein-specific challenges remain.
Screenings for proteins have typically been carried out by screening large numbers of random cDNA libraries. Traditional library screening techniques required the preparation of expressed proteins in phage vectors followed by immobilization of the protein on a membrane by a plaque lift procedure. This method is effective but is limited for several reasons. Clones do not always encode proteins in the correct reading frame and most proteins are not full length. Furthermore, in the bacterial expression system, abundant transcripts can often be overexpressed, while some proteins with low transcript number are not expressed at all. In addition, the bacterial system does not always permit the protein to be folded correctly.
High-throughput molecular biology techniques for identifying clone genes allow for DNA microarrays to study a variety of cloned genes. The genes can be attached to the surface of a support by physical or chemical means. The attached oligonucleotides may be in a random or pre-determined attachment orientation. For example, an oligonucleotide can be attached to support surface at the 5′- or 3′-end.
Certain aspects of microarray technology designed for oligonucleotides can be used to study proteins. The different physical and chemical properties of proteins as compared to oligonucleotides, however, requires new approaches. One key factor in producing protein microarrays is the mechanism for immobilizing and attaching proteins to a support. The method used should maintain protein function when function is being assayed, or protein structure when structure is assayed.
There is thus a need for means of attaching subject proteins to supports for use in a microarray. The present invention meets that need and more.